1. Field of the Invention
Affinity chromatography has become a valuable tool for separating biological materials, for example biologically active molecules such as small ligands, proteins, nucleic acids, enzymes, etc. In affinity chromatography, a substrate is immobilized on a granular support during the chromatography. By utilizing a column of said granular immobilized substrate, materials having affinity or binding specificity for the compounds bonded to the stationary phase can be separated from other materials in a mixture.
Typically, complex polysaccharide granules such as agarose beads, etc. are employed in affinity chromatography. The material of the granules may inherently have the desired sorption specifity or a suitable ligand may be bonded, often through so-called spacer-arms, to the granules by a variety of methods. Components in an ambient solution which have a high binding affinity for the materials of the granules or for the ligand will be preferentially bound to the granules. The bound components may be subsequently removed from the granules by contacting the latter with a solution which reduces the binding of the component for the material of the granules or for the ligand. Typically such desorption is accomplished by a substantial change in pH or ionic strength. Alternatively the sorbed component may be desorbed by another ligate which competes for the binding sites. Often chaotropic agents are used which by altering the secondary structure of the sorbed component effect desorption.
Examples of high specific ligands are:
Lictin absorbents for binding glycoproteins, glycolipids, polysaccharides, and related substances; PA1 Protein A from Staphylococcus aureus for binding many immunoglobulins; PA1 Cibacron.RTM. Blue F3G-A for binding albumin, interferons, growth factors, kinases and dehydrogenases; PA1 Monoclonal antibodies for binding biospecific antigens; PA1 Biospecific antigens for binding monocloncal antibodies and PA1 Hydrophobic groups (e.g. aliphatic or aromatic moieties) for binding proteins having hydrophobic regions.
Desorption of tightly bound, high molecular weight ligates is generally a rather slow process and in the presence of substantial changes in pH, ionic strength or high concentrations of chaotropic reagents may result in alterations in biological activity of the ligate or loss of biospecificity of the ligands.
It is an object of the present invention to provide processes and apparatuses which enable the comparatively rapid and economic recovery of bioactive ligates under comparatively gentle desorption conditions, thereby preserving a substantial fraction of the bio-activity.
These and other objects will become apparent from the following description of the invention.
2. Description of the Prior Art
(A) P. J. Brown et al (FEBS Letters, Vol. 83, No. 2, Nov. 1977 pp. 256-259) describe the absorption of antigen ligates on columns of granules having antibody ligands. Electrodes were subsequently placed at the top and bottom of the columns and a direct current passed for about one hour to remove the antigen by electrophoresis. The bioactivity of the recovered antigen was not reported.
(B) M. R. A. Morgan et al (J. Immun. Methods 23 (1978) pp. 381-387) describe sorption of immunoglobulin ligates in columns of beads in which the ligands were antisera to the immunoglobulins or Protein A from S aureus. The beads were placed on top of polyacrylamide disc gels. A direct current potential of 50 volts was applied for 4 to 5 hours and the ligates removed by electrophoresis. The bioactivity of the recovered sorbate was not reported.
(C) M. J. Igbal et. al. (FEBS Letters, Vol. 87, No. 2, March 1978, pp. 235-238) describe sorption of a hormone binding globulin by stirring with beads having ligands of androstanediol. The beads were transferred to a column having electrodes at either end. A direct current potential of 110 volts was applied for 5 hours which resulted in removal of the ligate. Substantial denaturation of the recovered ligate was reported.
(D) M. R. A. Morgan et al. (Analyt. Biochem. 105, pp. 1-5, 1980) describe the sorption of albumin in columns of beads having ligands of Cibacron.RTM. Blue F3G-A. The beads were subsequently placed on top of a column of polyacrylamide gel. Direct electric current was passed between electrodes located at the top and bottom of the column. The albumin was eluted electrophoretically. Elution times required to obtain substantial recoveries of albumin were 10 to 20 hours. The degree of denaturation of the albumin was not reported.